The involvement of Pseudomonas putida in basidiome initiation of the cultivated mushroom Agaricus bisporus.

Abstract

The involvement of pseudomonads In the process of basidiome initiation of the cultivated mushroom Agaricus bisporus was investigated. Pseudomonads used throughout the study were identified and Pseudomonas putida was shown to be responsible for initiating basidiome morphogenesis. The prodigious morphogenetical capabilities of a single P. putida and P. tolaasii colony was demonstrated and the production of rough colonial forms in peat casing soil was observed. Rough colonial forms of P. putida were found to be capable of promoting basidiome initiation. Cells of the rough colonial forms were better able to withstand nutrient limited conditions, were more resistant to UV light, produced greater amounts of siderophore and respired at a faster rate than the smooth colonial variants which indicates that they are stress tolerant forms. P. putida and P. tolaasii were shown to be positively attracted toward exudates of mushroom mycelium and adherence of these bacteria to hyphae was observed. Quantitative adherence studies, in conjunction with electron microscopy, revealed the ability of P. putida and P. tolaasii cells to adhere rapidly and firmly to hyphae. Differences in the chemotactic and adhesive abilities of smooth and rough colonial forms were observed. A new medium for the growth of A. bisporus was developed and a strain of A. bitorquis W19 which produces primordia in vitro, when grown in association with basidiome stimulatory pseudomonads, was used as a model system for assaying the effect of bacteria on basidiome initiation. Activated charcoal was shown to replace the effects of P. putida and fruit body initiation did not to occur in response to nutrient limitation. Non-living P. putida failed to promote basidiome initiation and iron, and Iron chelating agents did not stimulate fruiting: During the preliminary phase of the interaction between P. putida and A. bisporus, the bacterium was shown to markedly affect hyphal growth, colony morphology and the allocation of resources to the mycelium. Mutants of P. putida defective in their ability to promote basidiome initiation were produced by transposon Tn5 and UV light mutagenesis. The majority of these mutants were defective in their ability to uptake the ferric-siderophore complex implicating the involvement of iron regulated, membrane bound protein porins in the process of fruit body initiation. It is thought that the mushroom mycelium produces a 'self-Inhibitory-compound' which prevents basidiome morphogenesis until levels of this compound are reduced to below a threshold concentration. P. putida is thought facilitate this by Importing the Inhibitory substance into the cell, via the membrane bound protein porins, where it Is subsequently metabolized. A gene cloning and mapping system was developed In P. putida using RP4::mini-Mu. This plasmid was shown to promote transfer of auxotrophic markers in both homologous and heterologous matings and its potential as a gene cloning and mapping system in this organism was demonstrated.